Abstract

Objectives The aim of this study was to investigate, in a set of 93 mutation-negative long QT syndrome (LQTS) probands, the frequency of copy number variants (CNVs) in LQTS genes.

Background LQTS is an inherited cardiac arrhythmia characterized by a prolonged heart rate–corrected QT (QTc) interval associated with sudden cardiac death. Recent studies suggested the involvement of duplications or deletions in the occurrence of LQTS. However, their frequency remains unknown in LQTS patients.

Results We identified 3 different deletions in 3 unrelated families: 1 in KCNQ1 and 2 involving KCNH2. We showed in the largest family that the deletion involving KCNH2 is fully penetrant and segregates with the long QT phenotype in 7 affected members.

Conclusions Our study demonstrates that CNVs in KCNQ1 and KCNH2 explain around 3% of LQTS in patients with no point mutation in these genes. This percentage is likely higher than the frequency of point mutations in ANKB, KCNE1, KCNE2, KCNJ2, CACNA1C, CAV3, SCN4B, AKAP9, and SNTA1 together. Thus, we propose that CNV screening in KCNQ1 and KCNH2 may be performed routinely in LQTS patients.

Long QT syndrome (LQTS) is an inherited cardiac arrhythmia characterized by a prolonged heart rate–corrected QT (QTc) interval, which is associated with syncope and sudden death caused by torsades de pointes or polymorphic ventricular tachycardia. LQTS can be an autosomal recessive disorder (1), but the most common form is an autosomal dominant disorder called Romano-Ward syndrome (2,3). LQTS affects between 1 in 5,000 and 1 in 2,000 individuals (4,5). Molecular diagnosis is an important tool to guide diagnosis, treatment, and prevention strategies in LQTS patients. To date, more than 600 mutations (6) have been identified among 12 different genes: 5 genes encoding ion channel alpha subunits (KCNQ1 [7], KCNH2 [8], SCN5A [9], KCNJ2 [10], and CACNA1C [11]) and 7 genes encoding ion channel regulatory proteins (ANKB [12], KCNE1 [13], KCNE2 [14], CAV3 [15], SCN4B [16], AKAP9 [17], and SNTA1 [18]). In total, molecular diagnosis can resolve up to 70% of cases. More than 90% of those cases are due to mutations in KCNQ1, KCNH2, and SCN5A, corresponding to LQT1, LQT2, and LQT3, respectively (19,20). The lack of mutation detection in the remaining cases has been attributed to phenotyping errors, incomplete sensitivity of screening methods (denaturing high-performance liquid chromatography [dHPLC]) and direct sequencing), mutations in noncoding regions, or mutations in as of yet unknown genes.

Another source of negative molecular screening could be the presence of copy number variants (CNVs) affecting the major genes for LQTS, that would not be detectable using capillary sequencing. Interestingly, Bisgaard et al. (21) described in 2006 a large deletion (217 genes) including KCNH2 in a patient with mental retardation and a LQTS. In addition, Koopmann et al. (22) detected a 3.7-kb intragenic KCNH2 duplication in a Dutch family affected by LQTS. More recently, Eddy et al. (23) identified 2 deletions—1 in KCNQ1, a second in KCNH2—and a duplication in KCNH2 in LQTS patients. These different studies suggest that gene duplications or deletions can explain LQTS. However, the precise frequency of CNVs involving the main LQTS genes in patients with LQTS remains unknown. In this study, we investigated the involvement of rare deletions and duplications affecting KCNH2 and KCNQ1 in 93 probands with LQTS and in particular in 1 large family for which no putatively causative point mutations had been identified previously.

Methods

LQTS patients

This study was in agreement with the local guidelines for genetic research and has been approved by the local ethical committee. Two experts for rare arrhythmic diseases at the University Hospital of Nantes defined the LQTS phenotype by independent electrocardiogram (ECG) readings. Diagnosis of LQT syndrome was based on the QTc duration, the morphology of the T-wave, and the patient's clinical and family history. The Schwartz score has also been calculated for the 93 patients, and all of them have a Schwartz score of 3 or greater. QTc duration was calculated according to Bazett's formula. A prolongation of the QTc duration was defined as ≥440 ms for men (borderline between 430 and 439 ms) and as ≥460 ms for women (borderline between 450 and 459 ms) (24). Each patient underwent full medical examination to rule out syndromic forms of QT prolongation. Blood samples were collected after written informed consent. Mutations in coding regions and exon–intron boundaries for the 3 main LQTS-causing genes—KCNQ1, KCNH2, and SCN5A—were excluded by dHPLC or direct sequencing.

MLPA, QMPSF, and qPCR analyses

Multiplex Ligation-dependent Probe Amplification (MLPA) was performed using the SALSA P114 MLPA kit (MRC-Holland, Amsterdam, the Netherlands) and according to the MRC-Holland protocol (25). The SALSA P114 MLPA kit contains 20 probes interrogating the KCNQ1 gene, 9 for the KCNH2 gene, and 3 for the SCN5A gene. Abnormal profiles in MLPA analysis were completed with a locus-specific Quantitative Multiplex PCR of Short Fluorescent Fragment (QMPSF) (26) (see the supplemental Online Methods). Oligonucleotide complementary sequences to exons 5 and 15 of KCNH2 or exon 7, intron 7-8, and exon 8 of KCNQ1 were coamplified by PCR with an additional fragment, corresponding to exon 14 of MLH1, a gene located on chromosome 11 used as a control. The QMPSF conditions and primer sequences are available upon request. Quantitative PCR (qPCR) experiments were performed using the LightCycler 480 (Roche Molecular Systems, Mannheim, Germany) to validate genes with variable copy number. PCR reactions were prepared using the Power SYBR-Green PCR reagent kit (Applied Biosystems, Foster City, California) according to the manufacturer's protocol.

Linkage analysis

Two-point linkage analysis was performed with easy LINKAGE Plus software (version 5.02), by using an autosomal-dominant model of inheritance with complete penetrance and a disease allele frequency of 0.001 (T. Lindner, University of Würzburg, Würzburg, Germany).

Results

Ninety-three patients with LQTS were included in this study. No potentially causative point mutations in the KCNQ1, KCNH2, and SCN5A genes were identified by dHPLC or direct sequencing in any of these individuals. The patients (63% women) showed an average QTc duration of 556 ± 60 ms and an average age at diagnosis of 35 ± 22 years. Among the 93 patients, 33 (36%) have a Schwartz score of 3 and 60 (64%) show a Schwartz score of 4 or greater. Twenty-three patients (25%) had been resuscitated from sudden cardiac death, 58 (62%) had presented with a syncope, 30 (32%) with an arrhythmia event, and 15 (16%) had been implanted with an implantable cardioverter-defibrillator (ICD). The T-wave patterns for all patients were classified into 5 categories: normal T-wave morphology (n = 3, 3%), LQT1 (n = 11, 12%), LQT2 (n = 47, 51%), LQT3 (n = 13, 14%), and nonspecific (n = 19, 20%).

We screened for CNV involving the KCNQ1, KCNH2, and SCN5A genes in these 93 patients, by MLPA. We identified 1 heterozygote deletion in KCNQ1 and 2 heterozygote deletions involving KCNH2 in 3 unrelated patients. No CNVs were detected in SCN5A. Familial investigations were carried out for those 3 cases.

Family 1

Patient III:4 was diagnosed for LQTS after the occurrence of several episodes of torsades de pointes and ventricular fibrillation successfully resuscitated at age 23 years. Her ECG showed a prolonged QT interval (QTc = 554 ms) and a bifid T-wave strongly suggestive of LQT2 syndrome (Fig. 1). Because of the aborted cardiac arrest, she was implanted with an ICD. Her clinical examination found no other abnormalities. Familial recruitment led to the identification of 16 relatives through 3 generations (Fig. 1A). In addition to patient III:4, 6 family members had ECG abnormalities strongly suggestive of LQTS (Fig. 1B): patient I:2 (QTc = 465 ms), patient II:1 (QTc = 590 ms), patient II:7 (QTc = 615 ms), patient III:1 (QTc = 670 ms), patient III:5 (QTc = 518 ms), and patient III:6 (QTc = 610 ms). Each of these relatives was asymptomatic. ECGs performed in the other family members were normal (data not shown).

Detection of CNVs Involving the KCNH2 and KCNQ1 Genes in 3 Long QT Patients by MLPA

Each profile corresponds to relative peak ratios for control probes, KCNH2, KCNQ1, and SCN5A. The asterisks mark the detection of exonic deletions. (A)KCNH2/exons 4 to 14 are deleted in Family 1; (B)KCNH2/exons 1 to 14 are deleted in Family 2; (C)KCNQ1/exons 7 to 8 are deleted in Family 3. Y-axis represents the relative ratio of copy number. CNV = copy number variant; Ex = exon; Int = intron; MLPA = Multiplex Ligation-dependent Probe Amplification.

QMPSF was performed using 2 probes: the first in exon 5 confirmed the deletion, and the second in exon 15 demonstrated that the 3′ coding region of KCNH2 was also deleted (data not shown).

High-resolution array CGH analysis refined the size of the genomic rearrangement: the deletion, delimited by probes A_16_P18164054 (intron 3-4, centromeric breakpoint) to A_16_P01835269 (telomeric breakpoint), is 650 kb in length. Nineteen other genes are included in this rearrangement (including ABP1) (Fig. 3A), which maps to band 7q36.1.

(A) The proband from Family 1 carries a 650-kb-long deletion including KCNH2 and 19 other genes. (B) The proband in Family 3 carries a 145-kb-long deletion including KCNH2 and APB1. Black arrows indicate the clones flanking the deletion breakpoints. CGH = comparative genomic hybridization.

Interestingly, Redon et al. (27) identified a CNV in ABP1 downstream of the KCNH2 gene in individual NA12762 (chr7: 149, 976, 469–150, 101, 345) from the HapMap collection. We checked whether KCNH2 is encompassed by this CNV, using QMPSF on KCNH2 exons 5 and 15 and ABP1 exon 2. We found that the deletion identified by Redon et al. is limited to ABP1 and does not affect KCNH2 (data not shown).

The KCNH2 CNV was present in the 6 other affected family members (I:2, II:1, II:7, III:1, III:5, and III:6) but absent from 9 healthy members (Table 1). Linkage analysis was performed to evaluate segregation between the deletion and the LQT phenotype. Under the dominant model of inheritance, we obtained a maximum logarithm of the odds (LOD) score of 3.13 (θ = 0%).

Family 2

The proband is a woman diagnosed at age 28 years with LQTS after the occurrence of syncope triggered by acoustic stimulus (phone ringing). Her ECG showed a prolonged QT interval (QTc = 563 ms) and a bifid T-wave. ECGs were performed in the first-degree relatives: the mother, the brother, and the 2 sons (Fig. 4A). Prolongation of the QT interval was found only in the mother (I:2; QTc = 467 ms).

(A) Family 2 and (B) Family 3. The disease phenotype is transmitted as an autosomal-dominant trait. Open symbols depict unaffected members; solid symbols, long QT phenotypes; gray symbols, undetermined members; and question marks, unknown phenotypes. Circles indicate females; and squares, males. The probands are indicated by arrows. Genotypes are marked with +/− for heterozygous mutation and +/+ for wild type. QTc values (Bazett formula) are indicated below each individual. ECG are presented for affected members with ECG of arrhythmia when documented. Abbreviations as in Figure 1.

MLPA analysis showed a heterozygous deletion of KCNH2 (Fig. 2B). QMPSF confirmed the deletion of KCNH2 (probes in exons 5 and 15) and showed the deletion of ABP1 (probe in exon 2; data not shown). Array CGH analysis revealed that the CNV is 145 kb in length (probe A_16_P18165049 to probe A_16_P1835307) and includes entire copies of the KCNH2 and ABP1 genes (7q36.1) (Fig. 3B).

The same CNV was also found in 2 relatives: her affected mother (I:2) and her healthy brother (II:1) (Table 1).

Family 3

Patient II.1 was a girl diagnosed at age 14 years with the LQTS after the occurrence of an episode of syncope resulting from a stress with a temporary loss of hearing and tachycardia. Her ECG showed a prolonged QT interval (QTc = 490 ms) as well as a broad-base T-wave morphology on derivations V4 to V6. Her father presented with a T-wave pattern suggestive of LQT1 and a borderline QTc duration of 438 ms (Fig. 4B). Her mother and sister have normal T-wave morphology and QT interval duration (data not shown).

We identified by MLPA 1 heterozygote deletion that maps to band 11p15.5 and is restricted to KCNQ1 exons 7 and 8 in proband II:1 (Fig. 2C). The CNV was confirmed by 2 independent techniques: QMPSF with probes in exons 7 and 8, and qPCR with primers in exons 7 and 8 as well as intron 7-8 (data not shown). The CNV was inherited from the proband's father (I:1), whereas the proband's sister (II:2) did not carry the deletion (Fig. 4B, Table 1).

Discussion

CNV detection in LQTS genes

In this study, we evaluated the involvement of CNVs in KCNQ1, KCNH2, and SCN5A genes in patients affected by a typical form of LQTS, for whom previous molecular analysis had failed to identify point mutations in the known major LQTS genes. We detected 3 CNVs in 3 unrelated patients: 1 deletion in KCNQ1 and 2 deletions involving KCNH2.

Functional effect of the CNVs

The first deletion includes the exons 4 to 15 of KCNH2. This deletion could lead to a truncated protein. More probably, its effect may be similar to that of a premature termination codon mutation with subsequent nonsense-mediated decay degradation of the KCNH2 mRNA (28). Although this deletion includes 19 other genes, the patient presented with no abnormal phenotype other than prolonged QT duration.

The second deletion includes the whole KCNH2 gene, probably leading to haploinsufficiency and decreasing levels of the potassium current IKr (rapid components of the delayed rectifier potassium current) in ventricular cardiomyocytes.

The third deletion spans exons 7 to 8 of KCNQ1. Assuming that the deletion includes full copies of both exons, it leads to the lack of the second part of the p-loop (including GYGD motif), as well as the S6 transmembrane segment and 23 amino acids from the C-terminal part of the KvLQT1 channel subunit. Interestingly, previous studies have described splicing variants, involving deletion of either exon 7, exon 8, or both, leading to decreased IKs (slow components of the delayed rectifier potassium current) (29,30).

Family segregation analysis

We found perfect cosegregation between long QT phenotype and CNV inheritance in Family 1 (odds [LOD] score &gt;3), demonstrating that the KCNH2 deletion is responsible for this familial form of LQTS. One nonpenetrant patient (II:1) was identified in Family 2, in line with the observations of a previous study from others (31). In Family 3, the KCNQ1 deletion was found in 2 members presenting a T-wave pattern suggestive of LQT1. One of them presents with a prolonged QTc duration, the other with a borderline value (Fig. 4B).

Clinical implications

The 3 deletions identified are expected to lead to haploinsufficiency. Nonsense mutations or frameshift mutations brought about ≤50% reduction in cardiac repolarizing IKs or IKr potassium channel current. They are associated with a less severe phenotype than dominant-negative mutations but are known to cause sudden cardiac death (32). This less severe phenotype can explain the nonpenetrant case in Family 2 and the number of asymptomatic individuals. Furthermore, it is known that the nonpenetrant mutation carriers found in familial studies could experience syncope or cardiac arrest (33). However, more CNV studies are required to establish a genotype–phenotype correlation.

Indeed, our approach is focused on the exonic portions of the 3 most relevant genes involved in LQTS. We can expect that other functional and also nonfunctional CNVs may exist within the portions of the genes that have not been investigated (introns, regulatory elements, coding region not covered by the MLPA kit).

We note that similarly to that observed for the previously described point mutations, the T-wave morphologies observed in probands' ECGs are predictive for which LQTS gene is altered by CNV and thus remain a relevant indicator of the morbid gene (34).

Conclusions

In summary, our study demonstrates that CNVs involving major LQTS genes explain around 3% of additional LQTS cases (3 out of 93 patients, 95% confidence interval [CI]: 0.007 to 0.09) after failure of the classical molecular diagnostic screen (dHPLC and direct sequencing). This frequency corresponds to the percentage of CNVs versus point mutations already described in the published reports for a morbid gene such as BRCA1 (35). In our and others' experience, the frequency of CNV detection in KCNH2 and KCNQ1 is higher than the frequency of point mutations in ANKB, KCNE1, KCNE2, KCNJ2, CACNA1C, CAV3, SCN4B, AKAP9 and SNTA1 together (4,20). Thus, we conclude that CNV detection in the 2 major LQTS genes may be performed for patients diagnosed with LQTS when no point mutation has been detected by sequencing for KCNQ1, KCNH2, and SCN5A. In our experience (0 of 93 patients, 95% CI: 0 to 0.039) and from results of another study, CNVs involving SCN5A seems to be extremely rare in LQTS (36).

Our study demonstrates that genomic rearrangements in KCNQ1 and KCNH2 genes explain 3% of the LQTS cases in which no point mutation was found in the genes commonly involved. In consequence, we propose that screening for genomic rearrangement may be considered in the routine workup of LQTS in the absence of point mutations in the 3 major LQTS genes and before screening for point mutations in other LQTS genes.

Acknowledgments

The authors are greatly indebted to the patients and their families. They thank Christine Fruchet, Marion Chaventré, Christine Poulain, Patricia Bouillet, and Maïder Bessouet for assistance in the family screening; Anne Ponchaux, Jerome Buscail, Thierry Marsaud, Olivier Pichon, and Simon Lecointe for technical assistance; Fabrice Airaud for graphical representation matrix of MLPA data; and Dr. Stéphane Bézieau (Service de génétique médicale, Nantes) for his excellent work. The authors are also grateful to the Biogenouest platforms, in particular to Françoise Gros (plateforme de séquençage et de Génotypage), Catherine Chevalier, and Remi Houlgatte (plateforme transcriptome et puce à ADN) for their technical support.

Appendix

For supplemental methods, please see the online version of this article.

Appendix

Online Appendix

Screening for Copy Number Variation in Genes Associated With the Long QT Syndrome: Clinical Relevance

Toolbox

Thank you for your interest in spreading the word about JACC: Journal of the American College of CardiologyNOTE: We request your email address only as a reference for the recipient. We do not save email addresses.

Your Email *

Your Name *

Send To *

Enter multiple addresses on separate lines or separate them with commas.